Genetic testing

ABSTRACT

The present invention relates to an in vitro method for diagnosing or detecting a predisposition to a condition at least partially characterised by inappropriate fibrosis or scarring (e.g. Dupuytren&#39;s Disease). The method comprises examining the ZF9 gene, and regulatory elements thereof, derived from a subject of interest to detect the presence of a genetic poylmorphism or mutation in said gene which is linked with the condition.

[0001] The present invention relates to methods for the genetic testingof samples to determine the presence of polymorphisms in the Zf9 genethat are linked to a genetic predisposition to develop conditions atleast partially characterised by inappropriate Scarring or fibrosis.

[0002] A scar is an abnormal morphological structure resulting from aprevious injury or wound (e.g. an incision, excision or traunia). Scarsare composed of a connective tissue which is predominately a matrix ofcollagen types 1 and 3 and fibronectin. The scar may consist of collagenfibres in an abnormal organisation (as seen in normal scars of the skin)or it may be, an abnormal accumulation of connective tissue (as seen inscars of the central nervous system or pathological scarring of theskin).

[0003] Scarring is a usual consequence of the healing process in mostadult animal and human tissues. In the skin scars may be depressed belowthe surface or elevated above the surface of the skin. Hypertrophicscars are a more severe form of scarring that can arise in certainconditions or certain individuals. Hypertrophic scars are elevated abovethe normal surface of the skin and contain excessive collagen arrangedin an abnormal pattern. A keloid is a form of pathological scarringwhich is not only elevated above the surface of the skin but alsoextends beyond the boundaries of the original injury. In a keloid thereis excessive connective tissue which is organised in an abnormal fashionpredominantly in whorls of collagenous tissue.

[0004] There are believed to be genetic predispositions to forminginappropriate, and particularly pathological, scarring (e.g.hypertrophic scars and keloids). For instance, Africo-Carribean andMongoloid races are particularly prone to developing keloid scars.

[0005] There are numerous medical situations where scar formationrepresents a problem. Examples of such situations are scars of the skinwhere excessive scarring may be detrimental to tissue function andparticularly when scar contracture occurs (for instance skin burns andwounds that impair flexibility of a joint). The reduction of scarring tothe skin when cosmetic considerations are important is also highlydesirable. In the skin, hypertrophic or keloid scars particularly inAfrico-Caribbean and Mongoloid races) can cause functional and cosmeticimpairment and there is a need to prevent their occurrence. Scarringresulting from skin grafts in both donor sites and from the applicationof artificial skin can also be problematic and need to be minimised orprevented. Given the importance of scarring in such situations, it willbe appreciated that there is a need to be able to test a subject toinvestigate whether or not they will be susceptible to developinginappropriate scarring.

[0006] As well as scars of the skin, internal scarring or fibrosis canbe highly detrimental and specific examples include:

[0007] (i) Within the central nervous system, glial scarring can preventneuronal reconnection (e.g. following neuro-surgery or penetratinginjuries of the brain).

[0008] (ii) Scarring in the eye can be detrimental. In the cornea,scarring can result in abnormal opacity and lead to problems with visionor even blindness. In the retina, scarring can cause buckling or retinaldetachment and consequently blindness. Scarring following wound healingin operations to relieve pressure in glaucoma (e.g. glaucoma filtrationsurgery) results in the failure of the surgery whereby the aqueoushumour fails to drain and hence the glaucoma returns.

[0009] (iii) Scarring in the heart (e.g. following surgery or myocardialinfarction) can give rise to abnormal cardiac function.

[0010] (iv) Operations involving the abdomen or pelvis, often result inadhesion between viscera. For instance, adhesions between elements ofthe gut and the body wall may form and cause twisting in the bowel loopleading to ischaemia, gangrene and the necessity for emergency treatment(untreated they may even be fatal). Likewise, trauma or incisions to theguts can lead to scarring and scar contracture to strictures which causeocclusion of the lumen of the guts which again can be life threatening.

[0011] (v) Scarring in the pelvis in the region of the fallopian tubescan lead to infertility.

[0012] (vi) Scarring following injury to muscles can result in abnormalcontraction and hence poor muscular function.

[0013] (vii) Scarring or fibrosis following injury to tendons andligaments can result in serious loss of function.

[0014] Related to the above is the fact that there are a number ofmedical conditions known as fibrotic disorders in which the developmentof excessive fibrotic tissue leads to pathological derangement andmalfunctioning of tissue. Fibrotic disorders are characterised by theaccumulation of fibrous tissue (predominately collagens) in an abnormalfashion within the tissue. Accumulation of such fibrous tissues mayresult from a variety of disease processes. These diseases do notnecessarily have to be caused by surgery, traumatic injury or wounding.Fibrotic disorders are usually chronic. Examples of fibrotic disordersinclude cirrhosis of the liver, liver fibrosis, glomerulonephritis,pulmonary fibrosis, cystic fibrosis, scleroderma, myocardial fibrosis,fibrosis following myocardial infarction, central nervous systemfibrosis following a stroke or neuro-degenerative disorders (e.g.Alzheimer's Disease), proliferative vitreoretinopathy (PVR) andartluitis.

[0015] As is the case for scarring, it is believed that their is agenetic influence on the development of many fibrotic disorders or theseverity thereof. In some instances, the genetic influence may bedirectly responsible for the development of the disorder whereas forother fibrotic disorders there may be a genetic factor that influencesfibrotic development which is secondary to the primary cause of thedisorder (e.g. in cystic fibrosis).

[0016] One example of a disorder that involves fibrosis, and is alsobelieved to be influenced by genetics, is Dupuytren's disease (DD).

[0017] DD is a nodular palmar fibromatosis causing progressive andpermanent contracture of the digits. DD is often familial and is commonin individuals of Northern European extraction. In excess of 25% ofmales of Celtic races over 60 years of age have evidence of DD and it isconsidered to be one of the most common heritable disorders ofconnective tissue in Caucasians.

[0018] Autosomal dominance with variable penetrance has been proposed asthe likely mode of inheritance for DD. However no single gene has so farbeen identified in the literature as being responsible for thecondition. Furthermore it is unclear whether DD is a complex oligogeniccondition or a simple monogenic mendelian disorder. Accordingly theidentification of susceptible genetic loci would provide an idealapproach to unravelling the hereditary component of this common diseaseand would also be valuable in the subsequent development of treatmentand management strategies for DD.

[0019] It will be appreciated from the above that there is a need to beable to assess individuals for a susceptibility for developingconditions at least partially characterised by inappropriate scarring orfibrosis and also to assess the prognosis for an individual sufferingfrom such a condition. One way in which this may be approached is theidentification of polymorphisms or mutations in a gene that may beassociated with a particular medical condition. Once identified, suchpolymorphisms can provide useful information to physicians who need tomanage, treat or establish a susceptibility to developing a condition.One way in which the information may be used is in the development of agenetic test.

[0020] Genetic testing may be defined as the analytical testing of apatient's nucleic acid to determine if the DNA of a patient containsmutations (or polymorphisms) that either cause or increasesusceptibility to a condition or are in association with the genecausing the condition and are thus potentially indicative of apredisposition to that condition.

[0021] The early detection of a predisposition to a condition presentsthe best opportunity for medical intervention. Early geneticidentification of risk may improve the prognosis for a patient throughearly intervention before clinical symptoms manifest.

[0022] In cases where patients with similar symptoms are treated withvariable success with the same therapeutics, genetic testing maydifferentiate patients with a genetic rather than developmental basisfor their symptoms, thus leading to the potential need for differentapproaches to therapy.

[0023] It is an aim of the present invention to provide methods forgenetic screening to indicate a risk or predisposition to conditions atleast partially characterised by inappropriate fibrosis or scarring.

[0024] According to a first aspect of the present invention there isprovided an in vitro method for diagnosing or detecting a predispositionto a condition at least partially characterised by inappropriatefibrosis or scarring, the method comprising examining the ZF9 gene, andregulatory elements thereof, derived from a subject of interest todetect the presence of a genetic polymorphism or mutation.

[0025] By “polymorphism” we mean a region of a gene, or regulatingelements thereof, where the nucleotide base sequence may vary betweenindividuals. There is often a predominant genotype which represents theusual form, or wild type, of a gene with subsets of a population havinga polymorphism which confers a different genotype. Certain polymorphismscan be prevalent in individuals of particular ethnic backgrounds, orfrom specific geographical areas. A polymorphism may not affect functionof the gene; may lead to differences in the function of the gene; mayproduce an inactive gene product; or may modulate the production of thegene product.

[0026] By “mutation” we mean a region of a gene, or regulating elementsthereof, where the nucleotide base sequence may vary from the wild type.The mutation may for example comprise a base substitution, deletion oraddition. Certain mutant forms of the gene can be prevalent inindividuals of particular ethnic backgrounds, or from specificgeographical areas. A mutation may not affect function of the gene; maylead to differences in the function of the gene; may produce an inactivegene product; or may modulate the production of the gene product.

[0027] By “gene” we mean all coding sequences between the start and stopcodon of the Zf9 gene (including introns and exons).

[0028] By “regulatory elements” we mean the DNA that is 5′ and 3′ of thegene and which is involved in regulating gene transcription. Forinstance, transcription factor binding sequences, the TATA box, the 5′promoter and 5′ and 3′ untranslated regions (UTRs).

[0029] The method according to the first aspect of the invention allowsan investigator to identify a subject with a genetic polymorphism ormutation in the ZF9 gene or its regulatory elements to determine thosesubjects who have, or are more at risk of developing, a condition atleast partially characterised by inappropriate fibrosis or scarring.This allows for appropriate action to be taken to prevent or lessen thelikelihood of onset of the condition or to allow appropriate treatmentthereof. The method is also useful for establishing a prognosis for asubject that has already been diagnosed as suffering form a particularcondition.

[0030] The transcription factor Zf9 is one of a multitude of biologicalmolecules that is implicated in the pathophysiological processes thatlead to the development of a scar or fibrotic tissue. The gene for Zf9has been mapped to a locus on human chromosome 10p15. The sequence offull-length human (and rat) cDNAs has been identified by Ratziu et al.(Proc. Nat. Acad. Sci 95: 9500-9505, 1998) and the gene sequence of Zf9is publicly available as Gene Bank Accession No. AB017493.

[0031] Zf9 is also known as Core Promoter Element-Binding Protein (COPEBor CPBP); B Cell-derived Protooncogene 1 (BCDI); GC-Rich Sites BindingFactor (GBF); and Kmeppel-like Factor 6 (KLF6).

[0032] The inventors performed experiments to screen the Zf9 gene andits regulatory elements for polymorphisms and mutations which may beassociated with a condition at least partially characterised byinappropriate fibrosis or scarring. Having established such anassociation the inventors have established that DNA taken from a subjectmay be analysed to help establish a diagnosis of the condition or toestablish whether or not a subject is predisposed to develop such acondition.

[0033] The condition may be a form of pathological scarring of the skin(e.g. hypertrophic scarring or keloids) or an internal scar or fibrosisas mentioned above. Alternatively the condition may be a fibroticdisease or disorder also as mentioned above. Other conditions that maybe diagnosed or detected according to the method of the inventioninclude fibrotic disorders of the skin such as:

[0034] Sclerodemia

[0035] Systemic sclerosis

[0036] Crest Syndrome

[0037] Tuberous sclerosis with skin patches

[0038] Familial cutaneous collagenoma

[0039] Metabolic and immunologic disorders of the skin (porphyriacutanea tarda, chronic graft versus host disease)

[0040] Eosinophilic facsitis

[0041] Discoid lupus erythematosus, Dermatomyositis

[0042] Mixed connective tissue disease

[0043] Drug-induced skin fibrosis—bleomysin, PVC, silicates

[0044] Peyronies

[0045] Oral submucous fibrosis

[0046] Fibrosis induced following dietary and environmental exposures.

[0047] Fibrotic disorders of other organs may also be detected ordiagnosed. These include:

[0048] Pulmonary/cardiac fibrosis

[0049] Liver fibrosis/cirrhosis

[0050] Renal fibrosis

[0051] GI tract fibrosis

[0052] Drug induced fibrosis (e.g. post organ transplantation)

[0053] Central and peripheral nervous system fibrosis

[0054] Vascular system (veins and arteries) fibrosis

[0055] Male & female genitourinary tract fibrosis

[0056] Gynaecological (fallopian tube fibrosis, uterine fibromas)

[0057] The method of the first aspect of the invention is particularlysuited for diagnosing or detecting a predisposition to Dupuytren'sDisease.

[0058] The Zf9 gene to be examined is preferably derived from a humansubject. However it will be appreciated that DNA derived from animalsubjects of vetinary interest may also be tested according to the methodof the invention.

[0059] Preferred polymorphisms and mutations which may be detectedaccording to the first aspect of the invention are located in theuntranslated DNA 3′ of the Zf9 coding sequence. A most preferredpolymorphism is located at position 1140 of Gene Bank Accession No.AB017493460 (NCBI Assay Id (ss#) 20354; and Reference SNP Id (rs#)17731) and is referred to herein as “the 1140 polymorphism”.

[0060] This 1140 polymorphism represents a point mutation of a Guanosinenucleotide (G) to an Adenosine nucleotide (A). Genotypes AA, AG and GGwere identified for this polymorphism.

[0061] The point mutation is located 1022 bases from the start of thecoding sequence of the Zf9 gene and is found in the 3′ untranslatedregion. The two allelic forms have the following base sequence: The“G” form: gtccagggtcacccacataccatgcaccacgggtgctatgcc [G] cttcttacagg(Seq ID No. 1) acctttttagccctcaaaagaccttccaaggagaggccctggaggcaactgggtagggtgcagaaac The “A” form: gtccagggtcacccacataccatgcaccacgggtgctatgcc [A]cttcttacagga (Seq ID No. 2)cctttttagccctcaaaagaccttccaaggagaggccctggaggcaactgggtaggg tgcagaaac

[0062] Initial experiments established that the GG and AG genotype issignificantly over-represented in subjects with Dupuytren's Disease (DD)whereas a higher proportion of control subjects were of an AA genotype.

[0063] Further work has established that subjects with other fibroticdisorders (e.g. scleroderma or renal fibrosis) and subjects liable todevelop severe/pathological scarring (e.g. keloids) also have a highfrequency of the G allelle. Therefore the polymorphism may be examinedaccording to the method of the invention for diagnosing or detecting apredisposition to a variety of conditions at least partiallycharacterised by inappropriate fibrosis or scarring.

[0064] Various techniques may be used to detect polymorphisms ormutations according to the method of the invention.

[0065] A preferred technique involves Restriction Enzyme Digestion (RED)and is based upon the fact that polymorphisms can lead to the productionof different sized DNA fragments following treatment with a restrictionenzyme (because of the introduction or deletion of a restriction site bythe mutation causing the polymorphism). These fragments may bevisualised on gels and the polymorphism or mutant identified based uponthe number and size (i.e. distance moved on the gel) of the fragmentsfrom a DNA sample derived from a subject.

[0066] Preferably the DNA comprising the Zf9 gene and/or regulatoryelements thereof is amplified prior to detection of the polymorphismThis amplification is preferably by means of the polymerase chainreaction (PCR). For instance a preferred method according to theinvention known as the PCR-restriction fragment length polymorphismmethod (PCR-RFLP) is described in more detail in Example 1 and involvesPCR amplification of DNA containing the polymorphism prior to RED andsubsequent analysis.

[0067] PCR primers need to be designed such that they are suitable foramplifying a region around the relevant polymorphism. Suitable primersfor amplifying the 1140 polymorphism are listed below as SEQ ID No. 3and SEQ ID No. 4. Forward primer: 5′ GTCCAGGGTC ACCCACATAC 3′ (SEQ IDNo. 3) Reverse primer: 5′ GTTCTGCACC CTACCCAGTT 3′ (SEQ ID No. 4)

[0068] For some polymorphisms or mutations neither the wild type nor themutant allele abolishes or introduces a restriction enzyme site. Whenthis is the case, a restriction enzyme site may be introduced byspecifically designing PCR primers that introduce restriction sites intothe amplified product. The introduced enzyme site allows differentiationbetween polymorphic alleles and wild type by size analysis. For exampleif the restriction products of the amplified product are analysed by gelelectrophoresis (agarose or polyacrylamide gel, for example) the alleleswith the introduced restriction enzyme site will produce an extra bandon the gel.

[0069] Other techniques that may be used to detect polymorphisms ormutations according to the present invention include

[0070] (1) Direct sequencing of the polymorphic region of interest (e.g.using commercially available kits such as the Cy5™ Thermo Sequenase™ dyeterminator kit—Amersham Pharmacia Biotech);

[0071] (2) Sequence Specific Oligonucleotide Hybridization (SSO)(involving dot or slot blotting of amplified DNA molecules comprisingthe polymorphic region; hybridisation with labelled probes which aredesigned to be specific for each polymorphic variant; and detection ofsaid labels);

[0072] (3) Heteroduplex and single-stranded conformation polymorphism(SSCP) Analysis (involving analysis of electrophoresis band patterns ofdenatured amplified DNA molecules comprising the polymorphic region);

[0073] (4) Sequence Specific Priming (SSP) [also described asAmplification Refractory Mutation System (ARMS)];

[0074] (5) Mutation Scanning [e.g. using the PASSPORTS Mutation ScanningKit (Amersham Pharmacia Biotech)];

[0075] (6) Chemical Cleavage of Mismatch Analysis;

[0076] (7) Non-isotopic RNase Cleavage Assay (Ambion Ltd.);

[0077] (8) Enzyme Mismatch Cleavage Assay; and

[0078] (9) Single Nucleotide Extension Assay.

[0079] The method according to the first aspect of the invention isparticularly suitable for being carried out on genomic DNA, particularlyon isolated genomic DNA. Such genornic DNA may be isolated from blood ortissue samples (e.g. hair, oral buccal swabs, nail or skin) or fromother suitable sources using conventional methods. Preferably the DNA isisolated from whole blood or granulocytes.

[0080] A prediction or diagnosis based upon the method according to thepresent invention depends upon an association being made between aparticular condition and the specific polymorphism or mutant inquestion. Such associations were established by the inventors byperforming further experiments and making statistical analyses (e.g. seethe Example). Association between the 1140 polymorphism are discussedabove and in the Examples. Provision of data based upon associationanalyis enables a clinician to interpret the significance of genotypesidentified by sequencing DNA according to the method of the invention.The clinician may then make a judgment regarding the likelihood of apatient developing, or having, a particular disease or disorder. Suchknowledge is important in the clinical management of specific conditionsassociated with inappropriate scarring or fibrosis. It will beappreciated that data relating to the association of a particulargenotype with a condition may be provided to a user of the methodaccording to the invention (e.g. a technician or clinician) byincorporating a data sheet as part of a kit (see below).

[0081] Genetic testing may be carried out either pre-natally,peri-natally or post-natally when it is desired to test whether or not aneonate or child is likely to have inherited a predisposition to developa condition at least partially characterised by inappropriate fibrosisor scarring. This is particularly useful when there is believed to be afamily history of developing the condition.

[0082] The test is particularly useful for testing subjectspre-operatively. The results of such a test are useful for establishingwhether or not there could be healing complications for the subjectundergoing surgery (e.g. hypertrophic scarring, keloids or internalfibrosis/scarring).

[0083] The test is also useful before a therapeutic regimen isestablished for treating a condition characterised by inappropriatescarring or fibrosis. The results of the test according to the inventionmay be used by a clinician to help in the selection of medicaments usedand the dosage thereof.

[0084] It is most preferred that the method of the invention is used totest subjects with a family history of developing a condition at leastpartially characterised by inappropriate scarring or fibrosis.

[0085] The various elements required for a technician to perform themethod of the first aspect of the invention may be incorporated in to akit. Thus according to a second aspect of the present invention there isprovided a kit comprising:

[0086] A) PCR primers for amplifying genetic polymorphisms in the ZF9gene and regulatory elements thereof that are linked to a conditioncharacterised by inappropriate scaring or fibrosis; and

[0087] B) Control DNA samples of known genotype for each polymorphism.

[0088] It is preferred that the kit comprises primers specific for atarget sequence of sample DNA known to contain a polymorphism ofinterest For instance, suitable PCR primers for a kit for genotyping the1140 polymorphism are the primers of SEQ ID No. 3 and SEQ ID No. 4.

[0089] The kit may farther comprise:

[0090] C) a suitable restriction enzyme for generating fragments of theDNA sample;

[0091] D) a data card outlining linkage between a particularpolymorphism and a disease;

[0092] E) protocols for PCR amplification, restriction enzyme digestionof PCR products and agarose gel electrophoresis of DNA fragments;

[0093] F) relevant buffers.

[0094] Buffers provided with the Kit may be in liquid form andpreferably provided as pre-measured aliquots. Alternatively the buffersmay be in concentrated (or even powder form) for diluting.

[0095] The Kit may further comprise suitable reaction vessels,centrifuge tubes etc.

[0096] A multitude of biological molecules are involved in the fibroticor scarring process and it would be expected that many different factorslead to the development of a scar or fibrosis. The growth factor TGF-β1is an example of a biological molecule which has been implicated as amajor factor in the development of scars and fibrotic tissues. TheTGF-β1 gene is known to be polyniorphic and several polymorphisms of theTGF-β1 gene have been reported in the literature. The inventors exploredthe hypothesis that there is an association between four known TGF-β1polymorphisms and the fibrotic condition Dupuytren's disease. TGF-β1genotyping was performed in Caucasian individuals with DD and comparedwith a control Caucasian population These studies established that therewas a negative association between the Caucasian controls andDupuytren's disease cases for the TGF-β1 polymorphisms. The fact thatpolymorphisms in a fibrotic growth factor such as TGF-β1 did not exhibitany association with a condition characterised by inappropriate scarringor fibrosis, illustrates how surprising the inventors' discovery wasthat the polymorphisms in the gene for Zf9 showed such significantassociation.

[0097] The invention will be further described, by way of example only,with reference to the accompanying drawing, in which:

[0098]FIG. 1 is an electropherogram illustrating the polymorphism atposition 1140 Gene Accession No. AB017493; and

[0099]FIG. 2 is a photograph of a gel illustrating the difference insize of amplified DNA fragments corresponding to the genotypes GG, AGand AA in Example 1

EXAMPLE 1

[0100] Linkage Analysis of a ZF9 Polymorphism

[0101] Samples were taken from a group of patients with Dupuytren'sDisease (DD) and a control group to investigate the correlation betweena Zf9 polymorphism and the condition.

[0102] 1.1. Patients & Methods

[0103] 1.1.1 Patients

[0104] Dupuytren's patients (n=138) were entered into the study (119 menand 19 women). The age range was between 37 to 90 years with a mean ageof 55.8 years. Cases were all unrelated Caucasians from the Northwestregion of England, U.K. Dupuytren's cases were identified throughoperative record clinical codes from the South Manchester UniversityHospital Trust and Wrightington Hospital in the North West region. Allpatients were seen by a medically qualified person who took a fullmedical history using a proforma and examined both hands of each case.All cases had confirmed diagnosis of Dupuytren's disease pre-operativelywith the presence of characteristic dupuytren's nodules in the palm ofthe hand and/or digits with or without contracture of either the MCPJ orthe PIPJ.

[0105] A control group (n=161) comprised ethnically matched healthyCaucasian men and women and was selected from general practiceregisters.

[0106] The local and hospital ethical committees had given approval forthe study protocol and profomias. Written consent was obtained from allindividuals.

[0107] 1.1.2 DNA Extraction Method

[0108] Blood samples were collected from subjects using standardtechniques (15 ml of venous blood were collected).

[0109] DNA was extracted from peripheral blood cells using acommercially available DNA extraction kit (Qiagen,UK). DNAconcentrations were measured and diluted in buffer to 100 ng/μl usingsterile, Qiagen buffer.

[0110] 1.1.3 Genotyping Method

[0111] Zf9 genotyping was carried out using the polymerase chainreaction-restriction fragment length polymorphism (PCR-RFLP) method.

[0112] The polymorphism chosen for the study was found to be in the 3′untranslated region of the ZF9 gene and corresponded to the 1140polymorphism described above (NCBI Assay Id(ss#): 20354 Reference SNPId(rs#): 17731).

[0113] PCRs were carried out in 96 well microlitre plates. Each PCRconsisted of 1 μl of DNA (100 ng/μl ), 2.5 μl of x1 NH₄ buffer(Bioline), 2.5 μl of each 2 mM d'NTP (Behring), 0.1 μl of 0.1 unit Taqpolyrnerase (Bioline), 6 μl of Betaine, 0.75 μl MgCl₂, 0.1 μl each of2.5 pmol forward and reverse primer of Sequence I.D. No 2 and 3respectively and made up to 25 μl reaction mix with autoclaved,distilled water. The sequences of the primers used are given below:Forward GTCCAGGGTC ACCCACATAC; (Seq I.D. No. 3) Primer: and BackwardGTTCTGCACC CTACCCAGTT (Seq I.D. No. 4) Primer:

[0114] PCR was carried out under the following conditions: 2 minutes ofdenaturation at 95° C.; followed by 35 cycles of further denaturation of45 seconds at 95° C.; then 1 minute at either annealing temperature (aslisted in table 1), and 45 seconds of extension at 72° C.; a finalelongation step of 5 minutes at 72° C. was included.

[0115] The PCR-RFLP conditions was as follows: Amplified DNA (5 μl) wasdigested with the appropriate enzyme (0.8 μl) including buffer (1 μl)and made up to a 10 μl reaction mixture using distilled water. Digestionwas carried out overnight in a Hybaid Omnigene thermal cycler. Theenzyme used was Aci I (recognition sequence ccgc). Enzyme was purchasedfrom the New England Biolabs. The digested products were fractionated in4 % polyacrylamide gels and visualised by ethidium bromide andultraviolet light using standard procedures.

[0116] 1.1.4 Statistical Analysis

[0117] Association with DD was investigated by comparing thedistribution of its allele frequencies between DD patients and controlsusing a single global Pearson's chi-squared test. STATA 6 statisticaldata analysis programme was used to calculate p values and odds ratios.

[0118] 1.2 Results

[0119] The results of an electropherogram of the region surrounding the1140 region of Genbank Accession No. AB017493 are presented in FIG. 1and confirm the existence of the GIA polymorphism.

[0120] Having confirmed the existence of the polymorphism, DNA Fragmentsgenerated according to method 1.1.3 were run on polyacrylarnide gels asspecified. FIG. 2 illustrates that different genotypes generated DNAfragments of a unique size which could easily be differentiated byreading the gel. Accordingly gels were generated for each individual inboth the DD group and control group and statistical analysis made of anylinkage between genotype and health status.

[0121] Statistical analysis of the genotyped samples are provided inTable 1. These data illustrate that the GG and AG genotype issignificantly over-represented in subjects with Dupuytren's Disease (DD)whereas a higher proportion of control subjects were of an AA genotype.

[0122] Accordingly a linkage between the GG and AG genotypes of the 1140polymorphism in the 3′ untranslated region of the Zf9 gene and DD wasestablished. It will be appreciated that such a linkage will also existwith other conditions characterised by inappropriate scarring orfibrosis.

[0123] Table 1: Zf9 genotype & allele frequencies DD Cases CONTROL (n =138) (n = 161) Allele frequency 1 (A)  84 (30%) 128 (40%) 2 (G) 192(70%) 194 (60%) Genotype frequency 1 (A/A)  13 (9%)   23 (14%) 2 (A/G) 58 (42%)  82 (51%) 3 (G/G)  67 (49%)  56 (35%)

EXAMPLE 2

[0124] Having established a correlation between a conditioncharacterised by inappropriate scarring or fibrosis and a genotypeconferred by the 1140 polymorphism for ZF9, the methodology of Example 1was repeated to screen subjects according to the method of the firstaspect of the invention to establish the genotype of the subject.

[0125] The results of the genetic test could then be used by a clinicianto provide medical advice to the individuals. For instance, asymptomaticindividuals found to have the G allelle may be advised that they were atincreased risk of developing a condition characterised by inappropriatescarring or fibrosis. If appropriate, such individuals could be advisedto make life style changes and/or receive prophylactic treatment.Individuals having the G allelle, who are already suffering from acondition characterised by inappropriate scarring or fibrosis, may beadvised to adjust their medication or habits as they are potentially atrisk of developing a more severe form of the condition.

1 4 1 121 DNA Artificial Sequence Description of Artificial Sequenceprimer 1 gtccagggtc acccacatac catgcaccac gggtgctatg ccgcttcttacaggaccttt 60 ttagccctca aaagaccttc caaggagagg ccctggaggc aactgggtagggtgcagaaa 120 c 121 2 121 DNA Artificial Sequence Description ofArtificial Sequence primer 2 gtccagggtc acccacatac catgcaccac gggtgctatgccacttctta caggaccttt 60 ttagccctca aaagaccttc caaggagagg ccctggaggcaactgggtag ggtgcagaaa 120 c 121 3 20 DNA Artificial Sequence Descriptionof Artificial Sequence primer 3 gtccagggtc acccacatac 20 4 20 DNAArtificial Sequence Description of Artificial Sequence primer 4gttctgcacc ctacccagtt 20

1. An in vitro method for diagnosing or detecting a predisposition to acondition at least partially characterised by inappropriate fibrosis orscaring, the method comprising examining the Zf9 gene derived from asubject of interest to detect for the presence of a genetic polymorphismfrom Adenosine to Guanosine at a position 1022 bases 3′ of thetranscription start of the Zf9 gene.
 2. A method according to claim 1wherein the presence of a genetic polymorphism from Adenosine toGuanosine is examined in the region of DNA with the following nucleotidesequence:gtccagggtcacccacataccatgcaccacgggtgctatgccgcttcttacaggacctttttagccctca(Seq ID No. 1) aaagaccttccaaggagaggccctggaggcaactgggtagggtgcagaaac


3. The method according to claim 1 or 2 wherein the condition isDupuytren's Disease.
 4. The method according to claim 1 or 2 wherein thecondition is a keloid or hypertrophic scar.
 5. The method according toclaim 1 or 2 wherein the condition is any one of scleroderma; systemicsclerosis; crest syndrome; tuberous sclerosis with skin patches;familial cutaneous collagenoma; metabolic and immunologic disorders ofthe skin; eosinophilic facsitis; discoid lupus erythematosus;dermatomyositis; mixed connective tissue disease; drug-induced skinfibrosis: oral submucous fibrosis; fibrosis induced following dietaryand environmental exposures; pulmonary/cardiac fibrosis; liverfibrosis/cirrhosis; renal fibrosis; drug induced fibrosis; central andperipheral nervous system fibrosis; vascular system fibrosis; male andfemale genitourinary tract fibrosis; and gynaecological fibrosis.
 6. Themethod according to claim 1 or 2 wherein the condition is any one ofcirrhosis of the liver, liver fibrosis, glomerulonephritis, pulmonaryfibrosis, cystic fibrosis, scleroderma, myocardial fibrosis, fibrosisfollowing myocardial infarction, and central nervous system fibrosisfollowing a stroke or neuro-degenerative disorders
 7. A method accordingto any preceding claim wherein the ZF9 gene, and regulatory elementsthereof is derived from a sample of genomic DNA.
 8. A method accordingto claim 7 wherein the genomic DNA is isolated from blood or tissuesamples.
 9. A method according to any preceding claim wherein thepresence of a genetic polymorphism is determined following amplificationof at least a fragment of the DNA by polymerase chain reaction (PCR).10. A method according to claim 9 wherein the DNA is subjected to PCRamplification using the PCR primers: Forward primer: 5′ GTCCAGGGTCACCCACATAC 3′; (SEQ ID No. 3) and Reverse primer: 5′ GTTCTGCACCCTACCCAGTT 3′ (SEQ ID No. 4)


11. A method according to any preceding claim wherein the ZF9 gene, andregulatory elements thereof, is examined by restriction digestion andsize analysis.
 12. A kit, for use in the method according to any one ofclaims 9-11, comprising: A) a forward PCR primer and a reverse PCRprimer for amplifying a genetic polymorphism from Adenosine to Guanosineat a position 1022 bases 3′ of the transcription start of the Zf9 genewhich is linked to a condition characterised by inappropriate fibrosisor scarring; and B) Control DNA samples of known genotype for thepolymorphism.
 13. The kit according to claim 12 wherein the forward PCRprimer comprises 5′ GTCCAGGGTC ACCCACATAC 3′ (SEQ ID No. 3) and thereverse PCR primer comprises 5′ GTTCTGCACC CTACCCAGTT 3′ (SEQ ID No. 4)


14. The kit according to claim 11 or 12 wherein the kit furthercomprises a suitable restriction enzyme for generating fragments of DNA.15. The kit according to any one of claims 11 to 14 wherein the kitfurther comprises a data card outlining linkage between a particularpolymorphism and a condition and/or protocols for PCR amplification,restriction enzyme digestion of PCR products and agarose gelelectrophoresis of DNA fragments;
 16. The kit according to any one ofclaims 12-15 wherein the kit further comprises buffers for use in themethod according to any one of claim 9-11.